CD4+ T cells (TCD4+) play a critical role in combating most infections, are linked to a majority of autoimmune disorders, and are key components of anti-tumor immune responses. Unlike B cells, TCD4+ are triggered by contact with linear segments of antigen (termed epitopes) held at the surface of the antigen-bearing cell by major histocompatibility complex (MHC) class II molecules. The cellular mechanisms that lead to the exposure and binding of epitopes, and the subcellular locations where these events occur, remain poorly characterized. We have constructed a model that proposes two fundamental modes of epitope acquisition by class II molecules in separate subcellular compartments. Due to structural features of an antigen, some epitopes are revealed and loaded in early endosomes utilizing "mature" class II molecules that have cycled from the cell surface. In this benign environment, participation of the chaperonin DM is not required and antigens load onto class II in relatively intact form. Because most of the antigen does not contribute to binding, instead encouraging dissociation, such complexes are unstable at the cell surface. Due to structural constraints, other epitopes require the proteolytic activity of later endosomes to be revealed. Loading in such compartments involves nascent class II molecules and requires the participation of DM. Fragments delivered to the cell surface from this compartment are much shorter and, as a consequence, such epitopes are more stably expressed at the cell surface. Thus, targeting of proteins to different endocytic locations will influence the efficiency with which individual epitopes are presented. To test this model we will study the presentation of two H-2I-Ed- restricted epitopes derived from the influenza hemagglutinin (HA) molecule. By a number of criteria, "Site 3" (S3) is predicted to load in an early compartment while "Site 1" is predicted to load in a late compartment. In pursuing four specific aims we will rigorously test the model through the use of precise molecular manipulations, confocal immunomicroscopy, subcellular fractionation, and recombinant viral technology. There are several advantages to our antigen system including its clinical relevance, the extensive information available with regard to structure of HA, and the biological functions of HA as a receptor and acid-induced fusion protein. The work has the potential to contribute to a wide range of areas, from basic cell biology to vaccine design and in the long term may significantly enhance approaches to autoimmunity, cancer immunotherapy, and vaccine design.